Bottom Line:
This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio.This process preferentially contributed to LIP of memory cells.Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4(+) T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.

ABSTRACTLymphopenia-induced proliferation (LIP), a mechanism to maintain a constant number of T cells in circulation, occurs in both normal aging and autoimmune disease. The incidence of most autoimmune diseases increases with age, and premature CD4(+) T cell aging has been reported in several autoimmune diseases. In this study, we tested the hypothesis that premature CD4(+) T cell aging can cause autoimmune disease by examining whether premature CD4(+) T cell aging exists and causes LIP in our mouse model. Non-obese diabetic (NOD) mice were used because, in addition to Treg defects, the LIP of T cells has been shown to plays a causative role in the development of insulin-dependent diabetes mellitus (IDDM) in these mice. We found that with advancing age, NOD mice exhibited an accelerated decrease in the number of CD4(+) T cells due to the loss of naïve cells. This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio. In addition, both the percentage of CD28(+) cells in CD4(+) T cells and IL-2 production decreased, while the percentage of FAS(+)CD44(+) increased, suggesting that NOD mice exhibit premature CD4(+) T cell aging. This process preferentially contributed to LIP of memory cells. Therefore, our results suggest that premature CD4(+) T cell aging underlies the development of IDDM in NOD mice. Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4(+) T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.

pone-0089379-g005: Correlation between the percentage of CD4+ T cells that are FAS+CD44+ and age or the naïve/memory cell ratio.Splenocytes isolated from Balb/c and NOD mice were stained with PE-Cy5-conjugated anti-CD4, FITC-conjugated anti-CD44, and PE-conjugated anti-FAS antibodies. Gating was performed on live CD4+ T cells. (A, C) The dot plots represent the percentage of CD4+ T cells that are FAS+CD44+ in Balb/c mice (A) and NOD mice (C) versus age. (B, D) The dot plots represent the percentage of CD4+ T cells that are FAS+CD44+ in Balb/c mice (B) and NOD mice (D) versus naïve/memory ratio, as determined in Figure 2D. The experimental data were obtained from 2 pooled spleens for each experiment (Balb/c: n = 46 mice; NOD: n = 38 mice). Shown on the top is the gating strategy used in the analysis of the percentage of FAS+CD44+ cells in the CD4+ T cell population. Pearson correlation coefficients (rp), P values, and regression equations are shown in each panel.

Mentions:
In addition to molecules like CD28, which are important for T cell activation, molecules involved in T cell death also undergo age-related changes. The expression of FAS, a protein that triggers apoptosis when bound to its ligand, increases on human CD4+ T cells until approximately 61−74 years of age, and then decreases again [25]. The expression of CD45RO in humans or CD44 in mice is a marker for pre-activated/memory cells that also increases with age. The percentage of FAS+CD45RO+ T cells in CD4+ T cells exhibits the same trend as that observed in the total lymphocyte population [25]. In mice, the percentage of FAS+CD44+ cells in enriched splenic T cells is also higher in old (26 months) mice than in young (2 months) mice [26]. The percentage of FAS+CD44+ cells in splenic CD4+ T cells from Balb/c mice increased with age up to 91 weeks (Figure 5A) and was inversely correlated with the naïve/memory ratio (Figure 5B). In contrast, the percentage of FAS+CD44+ in splenic CD4+ T cells isolated from NOD mice (8−30 weeks of age) did not increase with advanced age (Figure 5C), probably because NOD mice undergo dynamic but inconsistent T cell aging. Therefore, we analyzed whether there is an inverse correlation between the percentage of FAS+CD44+ cells in splenic CD4+ T cells and the naïve/memory ratio of CD4+ T cells. Results of this analysis demonstrated that the percentage of FAS+CD44+ cells in splenic CD4+ T cells was inversely correlated with the naïve/memory ratio of CD4+ T cells in NOD mice (P = 0.04; Figure 5D). This result also indicated a dynamic T cell aging process in NOD mice.

pone-0089379-g005: Correlation between the percentage of CD4+ T cells that are FAS+CD44+ and age or the naïve/memory cell ratio.Splenocytes isolated from Balb/c and NOD mice were stained with PE-Cy5-conjugated anti-CD4, FITC-conjugated anti-CD44, and PE-conjugated anti-FAS antibodies. Gating was performed on live CD4+ T cells. (A, C) The dot plots represent the percentage of CD4+ T cells that are FAS+CD44+ in Balb/c mice (A) and NOD mice (C) versus age. (B, D) The dot plots represent the percentage of CD4+ T cells that are FAS+CD44+ in Balb/c mice (B) and NOD mice (D) versus naïve/memory ratio, as determined in Figure 2D. The experimental data were obtained from 2 pooled spleens for each experiment (Balb/c: n = 46 mice; NOD: n = 38 mice). Shown on the top is the gating strategy used in the analysis of the percentage of FAS+CD44+ cells in the CD4+ T cell population. Pearson correlation coefficients (rp), P values, and regression equations are shown in each panel.

Mentions:
In addition to molecules like CD28, which are important for T cell activation, molecules involved in T cell death also undergo age-related changes. The expression of FAS, a protein that triggers apoptosis when bound to its ligand, increases on human CD4+ T cells until approximately 61−74 years of age, and then decreases again [25]. The expression of CD45RO in humans or CD44 in mice is a marker for pre-activated/memory cells that also increases with age. The percentage of FAS+CD45RO+ T cells in CD4+ T cells exhibits the same trend as that observed in the total lymphocyte population [25]. In mice, the percentage of FAS+CD44+ cells in enriched splenic T cells is also higher in old (26 months) mice than in young (2 months) mice [26]. The percentage of FAS+CD44+ cells in splenic CD4+ T cells from Balb/c mice increased with age up to 91 weeks (Figure 5A) and was inversely correlated with the naïve/memory ratio (Figure 5B). In contrast, the percentage of FAS+CD44+ in splenic CD4+ T cells isolated from NOD mice (8−30 weeks of age) did not increase with advanced age (Figure 5C), probably because NOD mice undergo dynamic but inconsistent T cell aging. Therefore, we analyzed whether there is an inverse correlation between the percentage of FAS+CD44+ cells in splenic CD4+ T cells and the naïve/memory ratio of CD4+ T cells. Results of this analysis demonstrated that the percentage of FAS+CD44+ cells in splenic CD4+ T cells was inversely correlated with the naïve/memory ratio of CD4+ T cells in NOD mice (P = 0.04; Figure 5D). This result also indicated a dynamic T cell aging process in NOD mice.

Bottom Line:
This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio.This process preferentially contributed to LIP of memory cells.Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4(+) T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.

ABSTRACTLymphopenia-induced proliferation (LIP), a mechanism to maintain a constant number of T cells in circulation, occurs in both normal aging and autoimmune disease. The incidence of most autoimmune diseases increases with age, and premature CD4(+) T cell aging has been reported in several autoimmune diseases. In this study, we tested the hypothesis that premature CD4(+) T cell aging can cause autoimmune disease by examining whether premature CD4(+) T cell aging exists and causes LIP in our mouse model. Non-obese diabetic (NOD) mice were used because, in addition to Treg defects, the LIP of T cells has been shown to plays a causative role in the development of insulin-dependent diabetes mellitus (IDDM) in these mice. We found that with advancing age, NOD mice exhibited an accelerated decrease in the number of CD4(+) T cells due to the loss of naïve cells. This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio. In addition, both the percentage of CD28(+) cells in CD4(+) T cells and IL-2 production decreased, while the percentage of FAS(+)CD44(+) increased, suggesting that NOD mice exhibit premature CD4(+) T cell aging. This process preferentially contributed to LIP of memory cells. Therefore, our results suggest that premature CD4(+) T cell aging underlies the development of IDDM in NOD mice. Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4(+) T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.